Where materials, such as biological materials like proteins, have an isoelectric point, the materials can be separated by isoelectric focusing, wherein a solution is passed through an electric-potential field and the materials in the solution are allowed to gravitate toward the isoelectric point of the material in the field.
For example, when an electrical field is imposed upon a solution containing a mixture of proteins, the positively charged proteins migrate towards one electrode (the cathode) and the negatively charged proteins migrate in the opposite direction towards the other electrode (the anode). The characteristic charge of a particular protein, Z, is the net result of the hydrogen ion association/dissociation equilibria of the protein's numerous titratable groups; for example, --NH.sub.3.sup.+ =--NH.sub.2 +H.sup.+ and --COOH=--COO.sup.- +H.sup.+. Hence, Z is a function of pH, proteins are amphoteric and there is a characteristic pH value for each protein, pI, known as its isoelectric point, at which Z=0. Thus, if in addition to an electric field there is a gradient in the solution's pH, each protein can be made to migrate to a location between two electrodes at which the prevailing pH corresponds to that protein's pI. The vanishing of the protein's net charge at that point causes that particular protein species to cease migrating.
In conventional isoelectric focusing technology, the necessary pH gradient is created by adding to the protein solution a concentrated mixture of relatively low molecular weight buffers known as ampholytes, the pI values of which span the range of the proteins to be separated. The solution is contained between two electrodes, for example, some 10 cm apart. Electrolysis of water caused by the applied voltage generates a pH gradient which is stabilized by the relatively rapid isoelectric focusing of the ampholytes. This is followed in turn by the isoelectric focusing of the slower moving proteins, the concentrations of which are sufficiently lower than those of the ampholytes as not to perturb radically the pH gradient. This methodology has been applied both as an analytical tool; for example, in order to determine a particular proteins's pI value, and as a means of separation, leading, with further treatment, to the isolation of purified proteins.
Because of the low mobility of proteins, typically electrical fields of 100 to 1000 volts/cm are employed, and, even so, long time periods of one or more days are often required, in order to accomplish desired degrees of separation. Usually, cooling must be provided to offset appreciable resistive heat whcih might otherwise denature the proteins. Oxygen generated at the anode also may cause denaturation. An inherent disadvantage of the conventional methodology is the contamination of the proteins by the ampholytes used in the process.
Therefore, it is desirable to provide a new and improved method for the detection and/or separation of biological and other materials by isoelectric-focusing techniques, which overcome some of the disadvantages of the prior methods.